THE ROLE OF THE CTD PHOSPHATASE RTR1 AND POST-TRANSLATIONAL MODIFICATIONS IN REGULATION OF RNA POLYMERASE II Mary L. Cox Submitted to the faculty of the University Graduate School in partial fulfillment of the requirements for the degree Master of Science in the Department of Biochemistry and Molecular Biology Indiana University December 2013 ii Accepted by the Graduate Faculty, Indiana University, in partial fulfillment of the requirements for the degree of Master of Science. ____________________________________ Mark G. Goebl, PhD. Chair ____________________________________ Amber L. Mosley, PhD. Master’s Thesis Committee ____________________________________ Ronald C. Wek, PhD. iii ACKNOWLEDGEMENTS I would like to express my heartfelt appreciation to the following people; without whom, this work would not have been possible. You have all been true blessings in my life and I will be forever grateful.  To Dr. Amber Mosley- Thank you for inspiring me to be a better scientist, your endless patience, and friendship. You have gone above and beyond with your support, encouragement and mentoring. I am honored to have been a member of your lab and wish you success beyond measure.  To Dr. Mark Goebl- Thank you for your guidance and your efforts as a teacher in the Biotechnology Program and mentorship as the chairman of my thesis committee.  To Dr. Ron Wek- Thank you for your thoughtful participation as a member of my thesis committee. Your knowledge and advice have been extremely helpful and it has been a privilege to learn from you.  To Sharry Fears- Thank you for your tireless efforts in the laboratory portion of the Biotechnology courses. Your dependable preparation and knowledge of the material made difficult subject matter easier to understand.  To my colleagues in the Mosley Lab, past and present-Megan Zimmerly, Jerry Hunter, Melanie Fox, Michael Berna, Jason True, and Whitney Smith-Kinnaman- Thank you all for your knowledge and training with equipment, completion of experiments, encouragement, support and friendship. I have truly enjoyed knowing all of you and will miss our time together. iv  To my colleagues in the Biochemistry and Molecular Biology Department- Thank you for all your help with equipment and software loans, guidance, and advice: Dr. X. Charlie Dong and lab members, Dr. Timothy Corson and Kamakshi Shishtla, and Dr. Nuria Morral and lab members.  To my parents, Jack and Shirley: Thank you for instilling in me a sense of wonder about the world and a determination to explore it through education. Your unconditional love, support and encouragement have sustained me through this undertaking. My accomplishments are truly yours as well.  To my Awesome, Amazing, Incredible Husband, Yoda: “Thank you” is an insufficient sentiment for what you have sacrificed for this endeavor. Your unending kindness, patience, love and support mean the world to me. With each passing day, you are appreciated and loved beyond measure.  To my children: William, Catherine, Samantha, Alexander, Victoria, Amanda, and Cynthia- Thank you for giving me a life filled with great adventure and joy every day. Your love and patience through all the missed meals and activities is greatly appreciated. You are each extraordinary in your own way and I hope that this experience proves to you that there is nothing you can’t do if you have faith in yourself and are determined to succeed. v Mary L. Cox THE ROLE OF THE CTD PHOSPHATASE RTR1 AND POST-TRANSLATIONAL MODIFICATIONS IN REGULATION OF RNA POLYMERASE II RNA polymerase II (RNAPII) is regulated by multiple modifications to the C- terminal domain (CTD) of the largest subunit, Rpb1. This study has focused on the relationship between hyperphosphorylation of the CTD and RNAPII turnover and proteolytic degradation as well as post-translational modifications of the globular core of RNAPII. Following tandem affinity purification, western blot analysis showed that MG132 treated RTR1 ERG6 deletion yeast cells have accumulation of total RNAPII and in particular, the hyperphosphorylated form of the protein complex. In addition, proteomic studies using MuDPIT have revealed increased interaction between proteins of the ubiquitin-proteasome degradation system in the mutant MG132 treated yeast cells as well as potential ubiquitin and phosphorylation sites in RNAPII subunits, Rpb6 and Rpb1, respectively. A novel Rpb1 phosphorylation site, T1471-P, is located in the linker region between the CTD and globular domain of Rpb1 and will be the focus of future studies to determine biological significance of this post-translational modification. Mark G. Goebl, PhD. Chair vi TABLE OF CONTENTS LIST OF TABLES viii LIST OF FIGURES ix LIST OF ABBREVIATIONS xi INTRODUCTION I. The regulation of RNA Polymerase II transcription elongation 1 II. Post-translational modification of RNA Polymerase II 5 III. Overview of current known role of Rtr1 and other CTD Phosphatases 8 IV. Mechanisms for RNAPII degradation and recycling pathways 10 V. Proteasome inhibition and alternative approaches to study protein turnover 13 VI. Transformation of yeast for C-terminal domain tagging via homologous recombination 18 VII. Multidimensional protein identification technology (MuDPIT) to determine protein-protein interaction and potential post-translational modifications 21 MATERIALS AND METHODS I. Preparation of whole cell lysates following MG132 treatment 27 II. Western Blot Analysis 29 III. Transformation of S. cerevisiae deletion strains for C-terminal tagging for Rpb3 30 IV. Silver Staining Procedure 39 V. MG132 treatment of TAP-tagged S. cerevisiae mutant strains 40 VI. MuDPIT Analysis 41 VII. Malachite Green Phosphate Assay 44 vii RESULTS I. The effects of MG132 treatment on RNA Polymerase II phosphorylation in RTR1 deletion strains 45 II. Generation of S. cerevisiae strains Rpb3-TAP erg6∆and Rpb3-TAP erg6∆ rtr1∆ TAP tag through homologous recombination 49 III. Isolation of RNA Polymerase II complexes following MG132 treatment from wild-type, RTR1 deletion, ERG6 deletion, and RTR1/ERG6 double deletion strains 50 IV. Identification of ubiquitination sites in RNA Polymerase II purifications using a two-step bioinformatics analysis 56 V. Identification of phosphorylation sites in RNA Polymerase II purifications using a two-step bioinformatics analysis 67 VI. Can Threonine 1471 be dephophosphorylated by Rtr1 in vitro? 69 DISCUSSION 71 CONCLUSIONS 83 REFERENCES 85 CURRICULUM VITAE viii LIST OF TABLES 1. Summary of CTD kinases and phosphatases in S. cerevisiae and mammals 9 2. Selection of proteins identified by MuDPIT analysis of S. cerevisiae 15 3. Antibodies used for Western Blot analysis 30 4. Details of PCR reaction mixture 32 5. Details of PCR parameters 32 6. Comparison of peptide identifications between Proteome Discover/SEQUEST and Scaffold/X!Tandem 57 7. Proteins identified using Scaffold analysis for untreated Rpb3-TAP erg6Δ 60 8. Proteins identified using Scaffold analysis for MG132 treated Rpb3-TAP erg6Δ 61 9. Proteins identified using Scaffold analysis for untreated Rpb3-TAP erg6Δ rtr1Δ 62 10. Proteins identified using Scaffold analysis for MG132 treated Rpb3-TAP erg6Δrtr1Δ 63 11. Summary comparison of detected proteins from MS/MS data 78 ix LIST OF FIGURES 1. PDB-Viewer/Pov Ray generated model of complete ribbon structure for RNAPII complex. 2 2. Model of regulation of phosphorylation of the RNA Polymerase II CTD during transcription elongation in S. cerevisiae 8 3. RNAPII naturally pauses during transcription elongation 14 4. Current working model for RNAPII recycling and degradation following the loss of CTD phosphatases 16 5. Structure and sequence of the TAP tag and Schematic representation of TAP strategy 20 6. Schematic of TAP tag disruption cassette used to create Rpb3-TAP S. cerevisiae strains 21 7. Comparison of RNAPII Serine 5 phosphorylation using Western blot of four strains of S. cerevisiae both MG132 treated and untreated 46 8. Comparison of RNAPII Tyrosine 1 phosphorylation using Western blot analysis of MG132 treated and untreated yeast strains 47 9. Comparison of RNAPII CTD Serine 2, 5, and 7 phosphorylation Western blot analysis of MG132 treated and untreated yeast strains 48 10. Western blot analysis of Anti-CBP in yeast transformants isolated from YNB URA- agar after treatment with LiOAc and pBS1539 50 11. Silver Stain results of TAP-tagged deletion strains of S. cerevisiae 51 12. Growth curve comparison of Rpb3-TAP erg6Δ (aka erg6D) vs Rpb3-TAP erg6Δ rtr1Δ (aka erg6D-rtr1D). 52 13. Representative silver stains comparing yeast lysis methods prior to TAP procedure 53 14. Silver stain results from MG132 treated and untreated TAP tagged deletion strains 54 15. Comparison of elutions 1 and 2 from MG132 treated and untreated TAP tagged deletion strains as detected by Silver Stain 55 16. Peptide sequence coverage of RNAPIIs second largest subunit Rpb2 obtained from erg6Δ rtr1Δin the presence of MG132 using Scaffold software 66 x 17. Peptide sequence coverage and ion fragmentation spectra of RNAPIIs subunit Rpb6 obtained from erg6Δrtr1Δ in the presence of MG132 using Scaffold software. 67 18. Sequence coverage of Rpb1 obtained following erg6Δ rtr1Δin the presence of MG132 using Scaffold software. 68 19. Ion fragmentation spectra obtained from LC-MS/MS for erg6Δ rtr1Δin the presence of MG132 using Scaffold software 69 20. Malachite Green Phosphate Assay using recombinant GST-Rtr1 for potential substrate 70 [...]... polymerase II complex S2 Serine 2 of yeast RNA polymerase II C-terminal domain S2-P Phosphorylated serine 2 of yeast RNA polymerase II C-terminal domain S5 Serine 5 of yeast RNA polymerase II C-terminal domain S5-P Phosphorylated serine 5 of yeast RNA polymerase II C-terminal domain S7 Serine 7 of yeast RNA polymerase II C-terminal domain S7-P Phosphorylated serine 7 of yeast RNA polymerase II C-terminal... process the lengthening mRNA at the 5’ end As the RNAPII moves along the transcribed region of the gene, the shift of the CTD phosphorylated at serine 5 to serine 2 (S2-P) occurs through the activity of a cyclin-CDK kinase complex known as CTDK-1 (containing the cyclin dependent kinase subunit Ctk1) Consequently, at the 3’ end of the transcribed region the CTD is populated predominantly with S2-P CTD at... on phosphatase activity The specific roles of these phosphatases will be investigated further in this present study In order to test this model it is important to first understand the existing protein kinase and phosphatase activities Figure 2 Model of regulation of phosphorylation of the RNA Polymerase II CTD during transcription elongation in S cerevisiae The roles of the three known protein phosphatases... strains with the inability to form initiation competent RNAPII, leads to a modification in our working model (Figure 4) and our hypothesis that the loss of RNAPII recycling is observed in vivo in CTD phosphatase mutant strains for rtr1 , fcp1Δ, and ssu72Δ With a loss of the ability to reinitiate transcription and the potential for increased RNAPII stalling, it is possible that hyperphosphorylated RNAPII... heptad repeats (i.e CTD with S2-P and S5-P) In this thesis we will study the RNAPII modifications on both the CTD and the globular core of the enzyme in the presence and absence of Rtr1 IV Mechanisms for RNAPII degradation and recycling pathways RNAPII recycling is necessary to enable continuous transcription cycles in actively growing eukaryotic cells Actively transcribing RNAPII must overcome many... modifications that occur following deletion of the CTD phosphatase, RTR1 Finally, MuDPIT analysis will also be used to determine if additional interactions with specific factors can be identified that may add insight to potential mechanisms for RNA recycling III Overview of current known role of Rtr1 and other CTD Phosphatases Figure 2 shows our current working model of the role of RNAPII CTD phosphorylation... C-terminus of Rpb3 polymerase subunit at its endogenous locus (Washburn, 2008) Purification using Tandem Affinity Purification (TAP) of both erg6Δ and erg6 rtr1 could reveal novel interactions involved in signaling for proteasome degradation There are several alternatives to using proteasome inhibition to study the role of CTD phosphatases in regulation of RNA Polymerase II recycling For example, the use of. .. number of biochemical activities including: suppression of promoter-proximal stalling of RNAPII, promoter DNA melting, RNA Polymerase II loading onto the template strand, and stimulation of productive transcription (Cramer, 2004; Hirose & Ohkuma, 2007; Hsin & Manley, 2012) Specifically, immediately following RNAPII binding to the promoter, the CTD is phosphorylated at the S5 residue by a TFIIH-associated... are regulated by the phosphorylation state of the RNAPII CTD The creation of initiation competent RNAPII following a completed transcription cycle is ultimately dependent upon the activities of the three characterized CTD phosphatases since hyperphosphorylation of the CTD of Rpb1 has been shown to reduce RNAPII occupancy on active genes in the event of CTD 10 phosphatase defects (Gilmore & Washburn,... related WT Wild-type xiii INTRODUCTION I The regulation of RNA Polymerase II transcription elongation Transcription of DNA into cellular RNAs is accomplished by one of three highly conserved enzymes in higher eukaryotes (Vannini & Cramer, 2012) In eukaryotes, RNA polymerase II (RNAPII) is primarily responsible for transcribing DNA into messenger RNA (mRNA) as well as small nuclear RNA (snRNA) (Corden, Cadena, . Octylphenoxypolyethoxyethanol xii LiOAc Lithium Acetate Mfr Manufacturer MRM Multiple reaction monitor mRNA Messenger RNA MS Mass spectrometry/mass spectrometer MuDPIT Multidimensional protein identification. Biotechnology Program and mentorship as the chairman of my thesis committee.  To Dr. Ron Wek- Thank you for your thoughtful participation as a member of my thesis committee. Your knowledge. 11. Summary comparison of detected proteins from MS/MS data 78 ix LIST OF FIGURES 1. PDB-Viewer/Pov Ray generated model of complete ribbon structure for RNAPII complex. 2 2. Model